The objective of this proposal is to identify the signaling and molecular mechanisms whereby prolonged exposure to elevated fatty acids affects preproinsulin gene transcription, a phenomenon that contributes to the inexorable deterioration of beta-cell function in type 2 diabetes. Previously, we have demonstrated that palmitate inhibits insulin gene expression at the transcriptional level via ceramide synthesis in isolated islets. Specific Aim 1: To identify the signaling pathways mediating palmitate inhibition of insulin gene transcription in isolated islets and insulin-secreting cells. We will assess the involvement of the MAPK and PI3 kinase pathways by measuring kinase activity and phosphorylation state in response to glucose and palmitate. We will attempt to modulate palmitate-inhibition of transcription factor activity and insulin gene transcription by using pharmacological inhibitors and adenovirus-mediated overexpression of kinase isoforms. Specific Aim 2: To determine the mechanisms whereby palmitate inhibits expression of MafA and nuclear localization of PDX-1 in isolated islets and insulin-secreting cells. We will ascertain whether palmitate affects MafA expression at the transcriptional or post-transcriptional level by measuring mRNA stability and using promoter-reporter constructs;determine whether palmitate affects PX-1 nuclear localization by immunohistochemistry;and examine binding of MafA and PDX-1 to the endogenous insulin gene promoter by chromatin immunoprecipitation assays. Specific Aim 3: To ascertain whether combined hyperlipidemia and hyperglycemia affect insulin gene transcription in islets from chronically infused rats and high-fat fed mice. We will ascertain whether expression and binding activity of PDX-1 and MafA;activity of the insulin gene promoter;and insulin mRNA levels are affected in islets isolated from 1) Wistar rats following a 24- and 72-h infusion of glucose and fatty acids, alone or in combination; and 2) high-fat fed C57BI/6J mice. This project has the potential to uncover the cellular and molecular mechanisms by which excessive levels of fatty acids adversely affect pancreatic beta-cell function and thereby contribute to the deterioration of glucose homeostasis during the course of type 2 dabetes. It will provide new therapeutic targets aimed at preserving insulin secretion in type 2 diabetes, a devastating disease that affects more than 18 million Americans.